Proteins within the EF-hand protein family exhibit different conformational responses to Ca(2+) binding. Calmodulin and other members of the EF-hand protein family undergo major changes in conformation upon binding Ca(2+). However, some EF-hand proteins, such as calbindin D9k (Clb), bind Ca(2+) without a significant change in conformation. Here, we investigate the effects of replacement of a leucine at position 39 of the N-terminal domain of calmodulin (N-Cam) with a phenylalanine derived from Clb. This variant is studied alone and in the context of other mutations that affect the conformational properties of N-Cam. Strikingly, the introduction of Phe39, which is distant from the calcium binding sites, leads to a significant enhancement of Ca(2+) binding affinity, even in the context of other mutations which trap the protein in the closed form. The results yield novel insights into the evolution of EF-hand proteins as calcium sensors versus calcium buffers.